> Martin Offterdinger (a8803349 at unet.univie.ac.at) wrote:
> : Hi everyone!
> :
> : I have problems with a special restriction enzyme NdeI.
> : I try to cut PCR products after RT PCR with restriction enzymes.
> : What I do normally is to precipitate the PCR products with
> : isopropanol, wash with 80% EtOH air dry and dissolve the pellet
> : in the digestion mixture and incubate for 2h . Afterwards I load the
> : digest directly onto an agarose gel. This procedure works very well,
> : if I use BamH I to digest and I get my products cut very well-
> : BUT IT DOES NOT WORK WITH NDE I.
I've had problem with cloning PCR products with Nde1 in the past and in
my case I guessed that it was due to not enough bases flanking the
restriction site (eg EcoR1 only needs a couple of flanking bases but
Nde1 needs lots (8?)).
My solution was to do TA cloning (someones kit) and then cut the Nde1
insert out of this vector and clone into my target vector. This worked
fine.
Hope this helps,
Mike